The detection of the presence or absence of (or quantity of) one or more target nucleic acids in a sample or samples containing one or more target sequences is commonly practiced. For example, the detection of cancer and many infectious diseases, such as AIDS and hepatitis, routinely includes screening biological samples for the presence or absence of diagnostic nucleic acid sequences. Also, detecting the presence or absence of nucleic acid sequences is often used in forensic science, paternity testing, genetic counseling, and organ transplantation.
The gold standard in nucleic acid sequencing is capillary electrophoresis employing labeled dideoxy-nucleotides. Recently, next generation sequencing approaches have been described, bearing the promise of increased speed, throughput, and accuracy, and lower cost. Certain of these approaches employ polymerase-mediated incorporation of reversible terminator compounds (see for example U.S. Pat. No. 6,664,079). Other next-generation sequencing approaches employ ligation-mediated strategies (see for example WO2006/084132). Trade-offs in speed, accuracy, and cost continue to plague next generation sequencing approaches. The present teachings combine the strengths of polymerase-mediated approaches with certain aspects of ligation-mediated approaches to provided improved methods of performing highly parallel next generation sequencing.